MOLECULAR BIOLOGY CORE DEFINITION The purpose of the Molecular Biology core is to facilitate the application of the tools of molecular genetics to the study of gastrointestinal peptides and their physiological functions. In all aspects, the Molecular Biology Core functions to provide essential services and serves as an educational resource to all Center Investigators. Although basic molecular techniques are widely available to Center investigators through commercial sources, there remain specific services that are best provided by pooled resources and by highly trained personnel such as those that can be provided by a Center. During the prior funding period, three Core programs were provided: the Transgenic Animal Program, the Viral Vector Program and the Gene Expression Program. The Molecular Biology Core has added a Microarray Gene Chip Program and has essentially updated the Gene Expression Program to provide education and training in those techniques that can be performed in the investigators own laboratory after receiving some theoretical and practical training. Since investigator needs have likely moved beyond basic gene expression approaches to involve such techniques as chromatin structure and analysis, this primarily education program has been renamed the Molecular Techniques Training Program. Investigators needing assistance with basic molecular techniques, e.g., quantitative RT-PCR and more sophisticated techniques, e.g., chromatin immunoprecipitation assays (ChIP), can receive training from the Core Director (Dr. Merchant) and her associate (Dr. Bai). The specific objectives of the four Molecular Biology Core Programs are as follows: 1. The purpose of the Transgenic Rodent Program is to facilitate the generation of mouse and rat models to study regulatory peptides in a physiologic context. Transgenic and embryonic (ES) cell technology will be available to all Center members through a centralized core facility at the University of Michigan Medical Center. The Program's specific objectives are as follows: a. To serve as an educational resource for all aspects of transgenic and ES cell research, from the design of DNA constructs to animal husbandry and analysis of phenotypes. b. To prepare transgenic mice and rats from cloned DNA provided by Center investigators. c. To generate mouse ES cell clones with targeted gene mutations in collaboration with Center investigators. d. To prepare ES cell chimeric mice by microinjection of ES cells into blastocyts. e. To provide equipment and reagents for transgenic and ES cell production. f. To offer technologies to assist in rodent colony management, including mouse strain cryopreservation and recovery, conversion of mice to SPF status by embryo transfer, and in vitro fertilization (IVF). 2. The purpose of the Viral Vector Program is to facilitate the use of viral vectors for in vitro and in vivo gene transfer into mammalian cells, with emphasis on adenoviral vectors. The Vector Program's specific objectives are as follows: a. To serve as an educational resource for the use of viral vectors for gene transfer. b. To train Center investigators in how to construct and grow viral stocks to use in their research. c. To provide access to vectors and facilities to construct a variety of viral vectors with emphasis on adenoviral vectors. d. To provide reagent volume amounts of adenoviral vectors to Center investigators. 3. The purpose of the Microarray Gene Chip Program is to assist Center scientists in the use of highthroughput RNA profiling and genotyping methods towards delineating the role of gut hormones in health and disease. The Program comprises a separate component within the existing Affymetrix and cDNA Microarray Core, which serves all investigators affiliated with the University of Michigan Central Campus and Medical School Campus, as well as several NIH-funded Centers. As a component of the Core, the objectives of the Microarray Gene Chip Program are as follows: a. To actively assist Center investigators with the design, conduct and analysis of gene expression profiling experiments using oligonucleotide- and cDNA-based microarray technology. These services include probe preparation, labeling, hybridization, washing, and data acquisition for microarray and GeneChip experiments. b. To provide expertise and assistance for the statistical analysis of microarray-acquired data, for handling large data sets, and for data-mining large data sets; c. To provide the means for the validation of microarray-acquired data using quantitative polymerase chain reaction technology. d. To offer tools for genomic studies, including a SNP haplotyping service and Affymetrix HuSNP GeneChip services. 4. The purpose of the Molecular Techniques Training Program is to provide Center investigators with the theory behind and access to practical training in molecular techniques, e.g., RNA interference and chromatin immunoprecipitation assays (ChIP). The Program's specific objectives are as follows: a. To serve as an educational resource for the application of gene expression techniques, e.g., electrophoretic mobility shift assays (EMSAs, gel shifts), DNA footprinting, DNA methylation, transfections, promoter construction and analysis, quantitative PCR, reporter gene assays, RNA interference, ChIP analysis. b. To train investigators in specialized techniques, e.g., DNA binding and ChIP assays. c. To review applications to the Microarray Program. d. To provide and maintain the equipment to analyze reporter gene assays.